a surface active benzodiazepine receptor ligand for potential probing membrane order of gaba ...

A Surface Active Benzodiazepine Receptor Ligand for Potential ProbingMembrane Order of GABAA-Receptor Surroundings

Anahı V. Turina,† Benjamın Caruso,† Gloria I. Yranzo,‡ Elizabeth L. Moyano,‡ and Marıa A. Perillo*,†

Biofısica-Quımica, Catedra de Quımica Biologica, Departamento de Quımica, Facultad de Ciencias Exactas, Fısicas y Naturales,and INFIQC-Departamento de Quımica Organica, Facultad de Ciencias Quımicas, Universidad Nacional de Cordoba. Av. VelezSarsfield 1611, 5016 Cordoba, Argentina. Received April 28, 2008; Revised Manuscript Received July 17, 2008

A conjugable analogue of the benzodiazepine 5-(2-hydroxiphenyl)-7-nitro-benzo[e][1,4]diazepin-2(3H)-one N1-substituted with an aliphatic chain (CNZ acyl derivative, CAd) was synthesized. CAd inhibited FNZ binding toGABAA-R with an inhibition binding constant Ki ) 176 nM and expanded a model membrane packed up to 13mN/m when penetrating from the aqueous phase. CAd exhibited surface activity with a collapse pressure π )18.8 mN/m and minimal molecular area Amin ) 49 Å2/molecule at the closest molecular packing, resulting in fulland nonideal mixing with a phospholipid in a monolayer up to a molar fraction x = 0.1, decreasing its surfacepotential and contributing with a dipole that pointed its positive end toward the air and reoriented at the interfaceupon compression. These findings suggested that CAd could be stabilized at the membrane-water interface withits CNZ moiety stacked at the GABAA-R while its acyl chain can be inserted into the membrane depth.

1. INTRODUCTION

The 1,4-benzodiazepin-2-ones (BZDs)1 are drugs widely usedas anxiolytics, hypnotics, and anticonvulsants. They act byenhancing the activity of the neurotransmitter γ-amino butyricacid (GABA) through an allosteric binding site at the integralmembrane receptor GABAA (GABAA-R) (1). This is a pen-tameric protein that acts as a ligand-gated chloride channel andis the site of action of a variety of pharmacologically importantdrugs including benzodiazepines (BZDs) (2).

Several nonspecific mechanisms are known to affect theconformation and activity of membrane-bound protein receptors,such as (i) the coupling between hydrophobic mismatch andcurvature stress (3), (ii) changes in the lateral stresses profile(the depth-dependent distribution of lateral stresses within themembrane) which affect the conformation equilibrium and theactivity of intrinsic proteins, the function of which involves astructural change accompanied by a depth-dependent variationin its cross-sectional area within the transmembrane domain (4),and (iii) the dipolar arrangement of the membrane which wasshown to affect significantly the insertion, folding, conformation,and activity of membrane proteins (5, 6). With GABA-R as anexample, studies on natural membranes support the hypothesisthat the allosteric modulation of monoterpenes (6, 7) anddetergents (8) on this receptor comprise effects caused by druginsertion or other sources of mechanical tension on the su-pramolecular organization of the receptor environment, through

the mechanisms described above. Electrophysiological (9) andbinding (10) experiments showed that the cholesterol content,a known buffering mechanism of membrane microsviscosity,affected the coupling between the binding sites for BZDs andthe other drugs that interact with GABAA-R. Moreover, tem-perature-induced variations in membrane microviscosity alsomodulated the ligand binding to GABAA-R ((11) and refstherein).

Membrane microviscosity is considered the main modulatorymechanism of membrane protein function. Changes in thisgeneral membrane property are accompanied by modificationsin the hydrophobic thickness or in the lateral pressure profileof bilayers, which, as stated above, are indirect ways ofconnecting the protein structure with its function (12-17). It isknown that the plasma membrane of cells is substantiallyordered (e.g., RBL-2H3 cells (18)). It exhibits a limitinganisotropy (0.225 ( 0.005) significantly higher than thatobserved in the fluid phase of liposome bilayers. This indicatesthat, on average, a considerable amount of order exists in plasmamembrane (19). However, far from homogeneous, many typesof experiments have shown that, at the mesoscopic level, theplasma membranes of cells are patchy and locally differentiatedinto domains of different degrees of molecular order, some ofwhich arise through lipid-lipid interactions (20). For this reason,typical evaluation of microviscosity through the analysis ofgeneral anisotropy is not enough to understand the GABAA-Rsensitivity to membrane mechanical properties. The latter shouldinclude a scan of GABAA-R closest membrane surroundingsand may be achieved by the use of molecular probes that canremain close to the protein and, at the same time, allow thescanning of the local molecular organization within the mem-brane depth. Hence, we propose to use a probe bearing, at oneend, a GABAA-R ligand that will make the whole probe capableof stacking at the receptor and, at the other end, a hydrophobictail with a fluorescent or spin label moiety covalently attached.The latter, once inserted in the membrane, would providespectroscopic information about the molecular order within themembrane depth where it is located. This approach is based onsimilar studies on the nicotinic acetylcholine receptor (21). Inour laboratory, we have tackled this challenge in two stages.

* Corresponding author. Biofısica-Quımica, Departamento de Quım-ica, Facultad de Ciencias Exactas, Fısicas y Naturales, UniversidadNacional de Cordoba, Av. Velez Sarsfield 1611, 5016 Cordoba,Argentina. E-mail: [email protected]. Phone: +54-351-4344983 int5. FAX: +54-351-4334139.

† Departamento de Quımica.‡ INFIQC-Departamento de Quımica Organica.1 Abbreviations: BZD, benzodiazepines; CAd, clonazepam acyl

derivative; CNZ, clonazepam; dpPC, dipalmitoylphosphatidylcholine;DZ, diazepam; FNZ, flunitrazepam; GABA, gamma aminobutyric acid;GABAA-R, GABAA receptor; MGd, methyl glycinate derivative; NMR,nuclear magnetic resonance spectroscopy; sem, standard error of themean; SM, synaptosomal membranes; TLC, thin layer chromatography;TMS, tetramethyl silane.

Bioconjugate Chem. 2008, 19, 1888–18951888

10.1021/bc800175z CCC: $40.75 2008 American Chemical SocietyPublished on Web 08/13/2008

The first stage, consisting of the synthesis of a surface activeGABAA-R ligand, will be followed by the addition of thesensing group in a second stage.

In the present paper, we describe the synthesis, the GABAA-Rbinding kinetics, and the biophysical properties of a 5-(2-hydroxiphenyl)-7-nitro-benzo[e][1,4]diazepin-2(3H)-one (clon-azepam, CNZ) substituted at the N1 position with an aliphaticchain which was named CNZ acyl derivative (CAd). The abilityof this compound to interact and stabilize at the membrane-waterinterface was studied in monomolecular layers at the air-waterinterface by the Wilhelmy plate method. Molecular parametersdetermined from surface pressure-area isotherms were usedto predict not only the type of self-assembled structures thatCAd would be able to form in aqueous dispersions, but alsothe molecular features that would contribute to enhancing thestability of this compound in the typical planar configurationthat characterizes a membrane bilayer.

2. EXPERIMENTAL PROCEDURES

2.1. Materials. BZDs diazepam and clonazepam (DZ andCNZ) were kindly supplied by Products La Roche (Cordoba,Argentina). [3H]-FNZ was purchased from New EnglandNuclear Chemistry (E.I. DuPont de Nemours & Co. Inc., Boston,MA). Dipalmitoylphosphatidylcholine was obtained from AvantiPolar Lipids (Alabaster, AL). Other drugs and solvents were ofanalytical grade. Merck silica gel 60 was used for filtration andAldrich silica gel 60 F254 was used for preparative TLC.

2.2. Procedure for Preparation of CNZ Derivative. Amethanolic solution of sodium methoxide (1.85 mmol/mLmethanol) was added to a solution of CNZ (1.52 mmol) in 25mL of anhydrous methanol as described in ref 22. The mixturewas refluxed for a period of 15 min. After this time, 2.26 mmolof 1-bromooctane was added and subsequently refluxed for 12 h.After cooling, methanol was evaporated and the crude wasfiltered using silica gel. This mixture was then purified bypreparative TLC using hexane/ethyl acetate (80:20) and petro-leum ether/ethyl acetate/ethanol (95:4:1) as eluents. The reactionyield was 50% compound 3 and 30% compound 4 as mainproducts. These derivatives will be named CAd and MGd,respectively (see Scheme 1).

5-(2-Chlorophenyl)-6-nitro-1-octyl-1,3-dihydro-2H-1,4-ben-zodiazepin-2-one (3, CAd). 1H NMR (200 MHz, CDl3): δ 0.85(t, J ) 6.6 Hz, 3H), 1.24 (s, 10H), 1.60 (m, 2H), 4.31 (m, 2H),4.37 (dd, J ) 11 and 228 Hz, 2H), 7.30-7.50 (m, 4H), 7.55 (d,J ) 9.1 Hz, 1H), 7.62-7.70 (m, 1H), 7.93 (d, J ) 2.6 Hz, 1H),

8.35 (dd, J ) 2.6 and 9.1 Hz, 1H). 13C (50.33 MHz, CDCl3):δ ) 14.7, 23.2, 27.6, 29.1, 29.8, 29.8, 32.4, 48.2, 57.8, 123.2,125.3, 126.6, 128.1, 131.0, 131.8, 132.4, 133.7, 137.8, 143.9,147.8, 168.6, 169.2.

Methyl-N-{(2-chlorophenyl)-[2-nitro-6-(octylamino)phenyl]-methylene}glycinate (4, MGd). 1H NMR (200 MHz, CDCl3): δ0.89 (t, J ) 6.6 Hz, 3H), 1.30 (s, 10H), 1.84 (q, J ) 7.3 Hz,2H), 3.37 (m, 2H), 3.76 (s, 3H), 4.04 (q, J ) 19 and 47 Hz,2H), 6.72 (d, J ) 9 Hz, 1H), 7.09-7.14 (m, 1H), 7.35-7.60(m, 4H), 7.71 (d, J ) 2.6 Hz, 1H), 8.11 (dd, J ) 4 and 10 Hz),11.08 (s, 1H). 13C (50.33 MHz, CDCl3): δ ) 14.1, 22.6, 27.3,29.2, 29.3, 31.8, 43.5, 51.9, 54.2, 110.4, 115.9, 127.6, 128.6,129.8, 130.3, 131.4, 133.8, 135.1, 154.4, 170.2, 170.6.

2.3. NMR Spectroscopy. All starting materials were com-mercially available and solvents were distilled and dried beforeuse. 1H and 13C NMR spectra were recorded on a Bruker FT-200 (1H at 200 MHz and 13C at 50.33 Hz) spectrometer usingCDCl3. Chemical shifts are reported in parts per million (ppm)downfield from TMS.

2.4. Monolayer Studies. 2.4.1. Surface Pressure-Areaand Surface Potential-Area Compression Isotherms. Mono-molecular layers were prepared and monitored essentiallyaccording to Perillo et al. (23). Experiments were performed atroom temperature. The surface pressure (π, Wilhelmy platemethod via a platinized-Pt plate), surface potential (∆V, vibratingplate method), and the area enclosing the monolayer (A) wereautomatically measured with a Minitrough II (KSV, Helsinki,Finland). The Teflon trough used had 24 075 mm2 total area.Bidistilled water (230 mL total volume) was used as subphase.Lipid monolayers were formed by spreading, on the air-waterinterface, between 30 and 80 µL of 1 mg/mL chloroform-methanol 2:1 of (a) the newly synthesized CNZ acyl derivative(CAd), (b) a pure saturated phospholipid (dipalmitoylphosphati-dylcholine, dpPC), or (c) a binary mixture dpPC-CAd at a molarfraction (x) varying between 0 and 1. The π-A and ∆V-Acompression isotherms were recorded continuously, at a com-pression rate of 5 mm/min. Isotherms shown resulted fromtypical experiments repeated at least twice.

2.4.2. Compressional Modulus and Critical Packing Param-eter Calculation. The compressional modulus (K) was calculatedaccording to eq 1.

K)-(Aπ)(δπδA)π

(1)

The critical packing parameter (PC) was calculated accordingto the Israelachvili theory (24) by eq 2.

Pc )V

a0 · lc(2)

The average molecular area (a0) was experimentally deter-mined from π-A isotherms, and optimal values for thehydrocarbon volume (V) and chain length (lc) were calculatedfrom eqs 3 and 4, according to Perillo et al. ((23) and refstherein):

V= (27.4+ 26.9n)nch (3)

lc = 1.5+ 1.265n (4)

where n and nch are the number of methylene groups in thehydrocarbon chain and the number of chains per molecule,respectively. The molecular area (a0) is a function of theinterfacial free energy; hence, it was assigned the actual meanmolecular areas (Mma) at specific values of surface pressuretaken from the π-A isotherm of each monolayer at the differentcompositions studied. Pc values for the binary dpPC-CAdmixtures were calculated from the weighted mean values of (V)and (lc) of individual components.

Scheme 1. Synthesis of the CNZ-Acyl Derivative (CAd)

GABAA Receptor Surroundings’ Sensor Development Bioconjugate Chem., Vol. 19, No. 9, 2008 1889

2.4.3. Penetration of CAd in Monomolecular Layers ofdpPC at the Air-Water Interface. The aim of this experimentwas to determine the maximum value of π that allowed drugpenetration in the monolayer (πcutoff). These experiments weredone in a circular Teflon trough (4.5 cm diameter and 0.5 cmdepth). Between 5 and 30 µL of a chloroform-methanol 2:1solution of phospholipid were spread on an aqueous surface(bidistilled water) and about 5 min were allowed for solventevaporation. Monolayers were prepared at constant surface areabut at different initial surface pressures (πi). The temporalvariation of π induced by the CAd penetration into themonolayer after the injection of an ethanolic solution of thederivative in the subphase was measured until reaching a plateau(πmax). The values of ∆π ) πmax - πi were plotted against πi

and a straight line was fitted to them. The πcutoff was determinedfrom the intersection of the regression line with the abscissaaxis.

2.5. Synaptosomal Membrane Preparations. Synaptosomalmembranes (SM) were obtained from bovine brain cerebralcortex. Meninges were eliminated, the cortex dissected, and theSM were purified essentially according to the method of Ennaand Snyder, modified by Perillo and Arce (25), lyophilized andstored at -20 °C. Immediately before use, membranes wereresuspended in 50 mM pH 7 Hepes buffer containing 100 mMNaCl at a final protein concentration of 0.25 mg/mL. This SMsuspension was used as membrane receptor preparation andGABAA-R source in the experiments that followed.

2.6. Competition Binding Experiments. The aim of thisexperiment was to assess the ability of the newly synthesizedCAd to displace [3H]-FNZ from its binding site at the GABAA-Rin SM.

Binding was performed essentially as described previously(26). The whole procedure was carried out at 4 °C. Theincubation system contained, in a final volume of 230 µL, theSM suspension at a final protein concentration of 0.25 mg/mL,3 nM (minimum specific activity 74.1 Ci/mmol) [3H]-FNZ, 100mM NaCl-50 mM Tris-HCl pH 7.4 buffer containing 9.4 µMDZ or 10 µM CAd (final concentrations). Samples wereincubated at 4 °C in the dark for 1 h and then filtered throughSS filters (Whatman GF/B type) with a Brandel automaticfiltration apparatus (Brandel, Gaithersburg, MD). After filterswere rinsed and dried in the air, they were placed in vialscontaining 2.5 mL of scintillation liquid (25% v/v Triton X-100,0.3% w/v diphenyloxazole in toluene). The retained radioactivitywas measured with a scintillation spectrometer Rackbeta 1214(Pharmacia-LKB, Finland) at 60% efficiency for tritium. Specificbinding (B) was calculated as the difference between totalbinding (TB) and nonspecific binding (NB) determined in theabsence and in the presence of 9 nM DZ, respectively. Proteinconcentration was determined by the method of Lowry (27).

2.7. Statistical Analysis. Binding data were statisticallyanalyzed using a two-tailed Student’s t-test for independentsamples. p < 0.05 was considered to be statistically significant.Regression analysis was done by the least-squares method (28).

3. RESULTS AND DISCUSSION

3.1. Synthesis of the Probe Precursor. The probe precursor(3, CAd) was synthesized from the sodium salt (2) of 5-(2-chlorophenyl)-7-nitro-1,3-dihydro-2H-1,4-benzodiazepin-2-one (CNZ) (1) and 1-bromooctane (Scheme 1). The salt (2) wasobtained by the treatment of CNZ with sodium methoxideaccording to the literature (22). The reaction of the BZD 1 andthe bromoderivative was carried out in situ without isolation ofthe salt intermediate. The methyl N-methylene glycinate 4(MGd) was also obtained in this reaction by the hydrolysisreaction of the benzodiazepinone ring in the presence ofmethanol. The formation of compound 4 could not be avoided

even when the reaction times and temperatures were modified.Therefore, compound 3 was purified as described in section 2.2(purity was higher than 95%). Detailed spectra are provided asSupporting Information.

3.2. [3H]-FNZ Displacement Experiments. The ability ofCAd to displace [3H]FNZ was evaluated by means of aradioreceptor binding assay. Results were analyzed in compari-son with the well-known competitive inhibitor DZ. The results,depicted in Figure 1, show that DZ as well as CAd at similarconcentrations (9 and 10 µM, respectively) were able to displace80% and 59% of the total [3H]FNZ bound to SM, respectively.According to the denifition given in the Experimental Proceduressection, the 20% residual binding in the presence of 9 nM DZcorresponded to nonspecific binding. Furthermore, CAd at theconcentration assayed left a 21% undisplaced binding over thenonspecific indicating a lower affinity for the GABAA-R withrespect to DZ.

Assuming that CAd induces a competitive inhibition of[3H]FNZ binding, the inhibition binding constant (Ki) of thisdisplacement agent can be calculated from eq 5 as follows:

B)BmaxL

Kd(1+[I]Ki

)+ L(5)

where L is the free radioligand concentration (3 nM), B andBmax are the specific activities of bound radioligand (expressedin moles per protein mass units) at L or at saturating radioligandconcentration, respectively, and Kd is the dissociation bindingconstant of the radioligand-receptor interaction.

With Bmax ) 1404 fmol/mg protein and Kd ) 2.28 nM for[3H]FNZ binding to SM (26) and the experimental data shownin Figure 1, the Ki values for CAd were 176 nM. This valuewas similar to previously reported data (29) for other BZDs.This was a strong indication that the CNZ acyl derivative notonly could interact with the BZD binding site at GABAA-R,but also that it was bound with an affinity comparable with thatof a typical BZD.

3.3. Monomolecular Layers at the Air-Water Interface.3.3.1. Surface Pressure-Molecular Area Isotherms. Surfacepressure-area isotherms are shown in Figure 2. In Figure 2a,an isotherm of pure dpPC is shown as a reference. Dashed linesindicate the determination of the collapse pressure (πc ) 55.5mN/m), the molecular area at the closest packing also knownas minimal molecular area (Amin ) 40 Å2), and the transitionsurface pressure (πT ) 6 mN/m) corresponding to the bidimen-sional phase transition characteristic of dpPC.

Figure 1. Displacement of the [3H]-FNZ bound at synaptosomalmembranes from bovine cortex induced by CAd and DZ at similarconcentrations. The [3H]-FNZ and protein concentration used were 3nM and 0.25 mg prot/mL, respectively. Other details were describedin the Experimental Procedures section. Data shown are the mean (sem of triplicates. *, significantly different with respect to E (p < 0.01,Student’s t test).

1890 Bioconjugate Chem., Vol. 19, No. 9, 2008 Turina et al.

CAd was able to form monomolecular layers at the air-waterinterface both alone (Figure 2a) as well as in mixtures with dpPCat various molar fractions (x) (Figure 2b). From the analysis ofthe π-A compression isotherm of CAd (Figure 2a), a collapsepressure πc ) 18.8 mN/m and Amin ) 49 Å2 were determinedand no bidimensional transitions were observed. In comparisonwith dpPC, CAd exhibited lower stability as reflected by itslower πc value, which may be due to the shorter length of itshydrocarbon chain. This provides a low London dispersionenergy stabilizing the aggregate, which is not enough tocounterbalance the repulsive interactions between the polar headgroups. This leads to an inefficient molecular packing explainingthe fact that, in spite of having only one hydrocarbon chainand not two as in the case of dpPC, CAd exhibits a minimalmolecular area substantially higher than that of the phospholipid.

The CAd/dpPC mixtures exhibited full miscibility up to xCAd

= 0.1. Above xCAd ) 0.1, mixtures showed several bidimen-sional reorganizations indicating the occurrence of partialcollapse processes of different bidimensional phases (enrichedin dpPC, within the range 0.1 < xCAd < 0.4, or in CAd, withinthe range 0.4 < xCAd ) 1) coexisting in the monolayer. Thisindicated a partial miscibility of both components (Figure 2b).Further analysis was done through a phase diagram (see Figure3 below).

The effect of the proportion of CAd in the mixtures withdpPC on the monolayer elasticity was analyzed through thecompressibility modulus K (Figure 2c) in the conditions of fullmiscibility at all compositions (6 mN/m) or compositionally

dependent miscibility (35 mN/m). At 6 mN/m, K increasedcontinuously as a function of xCAd reflecting both the highercoherence of CAd films with respect to the liquid expandedphase of dpPC (corresponding to xCAd ) 0) as well as fullmiscibility of both compounds at this lateral pressure. At 35mN/m, K decreased as a function of xCAd showing discontinuitiesthat reinforced the different miscibility regimes exhibited bythe phase diagram (miscibility of CAd in dpPC which is lost atxCAd e 0.1) and suggested a partial miscibility of dpPC in acontinuous CAd phase within the range 0.4 < xdpPC < 0.6).

3.3.2. Surface Electrostatics. The surface potential ∆V-areaisotherms (Figure 2d) showed that ∆V increased upon compres-sion and decreased as a function of CAd in the mixtures. Atthe closest packing, ∆VdpPC ) 530 mV and ∆VCAd ) 296 mV,while the mixtures showed intermediate values. Surface potentialis a measure of the electrostatic field gradient perpendicular tothe membrane interface and thus varies considerably with themolecular surface density and with changes in orientationaccompanying the monolayer compression. Taking the dielectricconstant of the medium (water) as unity, the molecular dipolemoment can be calculated according to eq 6

∆V) 12 × πA

µ⊥ +ψ0 (6)

where π ) 3.1416, A (molecular area), and V (surface potential)are expressed in Å2.molecule-1 and mV, respectively, µ⊥(expressed in Debye units, mD) is the apparent (resultant)perpendicular dipole moment of the molecule, and ψ0 is the

Figure 2. Behavior of dpPC/CAd binary mixtures at the air-water interface. (a) Collapse pressure (πc), transition pressure (πT), and minimalmolecular area (Amin) determination exemplified over a dpPC π-A compression isotherm. (b) Surface pressure-Mma isotherms of dpPC-CAdbinary mixtures. (c) Compressional modulus at 6 and 35 mN/m, as a function of monolayer composition. (d) Surface potential-Mma and (e)µ⊥ -Mma compression isotherms of dpPC-CAd binary mixtures. Dipole moments (µ⊥ ) calculated according to eq 6 from ∆V values taken frompanel (d) depicted as a function of Mma (f) Resultant dipolar modulus at 6 and 35 mN/m, as a function of monolayer composition. Numbers inpanel (e) indicate the CAd molar fraction.


electrostatic potential difference at the interface caused by theionic double layer in ionized monolayers (for unchargedmolecules ψ0 ) 0) (23, 30). The parameter µ⊥ contains differentelectrostatic contributions. In the case of uncharged moleculessuch as dpPC (a zwitterionic phospholipid) and CAd (unchargedat the pH assayed), main contributions arise from the resultantdipole moments of the monolayer components and those of thewater hydration network (water molecules oriented at the polarheadgroup). In turn, contributions from monolayer componentsinclude the polar headgroup and hydrocarbon chain contribu-tions. An initial increase in the magnitude of µ⊥ was observedupon compression due to molecular reorientations at the surface(Figure 2e). Beyond a maximum, all µ⊥ vs Mma plots showeda decreasing trend which, in conjunction with the informationtaken from the π-Mma curves, can be interpreted as anindication of a monolayer compositional change due to instabil-ity occurring at high surface pressures. Figure 2f shows that, atπ low enough to allow total mixing between dpPC and CAd,µ⊥ decreased upon an increase in xCAd. At a high surface pressure(35 mN/m), (a) within the xCAd range that allowed miscibility,µ⊥ was higher than at 6 mN/m, and (b) at xCAd > 0.1, µ⊥ valuesexhibited a decreasing tendency even more noticeable than at6 mN/m which, in this case, would be reflecting the partialcollapse of the monolayer (see phase diagram in Figure 3).

3.3.3. dpPC-CAd Binary Mixtures: Phase Diagram andPrediction of Their Self-Assembling Structures in Water. Fromdata shown in Figure 2b, the π-x phase diagram was con-structed (Figure 3). Lines in Figure 3 indicate the presence ofπ-x states at which phase transitions occur and delimit regionsof single bidimensional phases that may coexist with an alreadycollapsed phase. A single monolayer phase can be found at lowpressures within the whole compositional range. This monolayershows a πT that varies continuously with xCAd (triangles jointby a dotted line). This πT might be associated either with abidimensional phase transition or with a partial collapse of the

monolayer. The second hypothesis is supported by the shapeinspection of π-A isotherms at the high xCAd (e.g., above 0.6)which suggests that after a collapse point monolayers go throughovercompression states. At higher pressures, another partialcollapse is observed at a πc that remains invariant up to xCAd )0.4 and then, at xCAd > 0.4, it decreases continuously with xCAd

(hollow circles) up to the point where πc equaled the πc valueof pure CAd. Hence, CAd seems totally miscible in dpPC upto xCAd ) 0.1, exhibiting a bidimensional phase transitionbetween 6 and 8 mN/m. Within the xCAd range 0.1-0.2, thesolubility of CAd in dpPC would be reached. This is based onthe fact that, upon compression, the collapse pressure of mixtureswith 0.1 < xCAd < 0.2 decreased from 39 to 37 mN/m, while at0.1 < xCAd < 0.4, the πc value remained constant at πc ) 37mN/m. Within this compositional range, there was a remainingmonolayer that would be composed of a dpPC excess asindicated by a third collapse point which had the typical valueof 55 mN/m. The interfacial stability of mixtures with xCAd <0.4 decreased continuously as a function of the xCAd as indicatedby the decreasing πc. Moreover, within this compositional range,after the monolayer collapsed no excess of dpPC remained stableat the air-water interface.

Critical packing parameter values (Pc) allow the predictionof the type of self-assembling structures that would be formedwhen an amphipathic substance is dispersed in water. Pc valuesfor the binary dpPC-CAd mixtures were calculated by eqs 2, 3,and 4 using values of mean molecular area which were takenfrom the isotherms shown in Figure 2b. Those Pc values weresuperimposed on the phase diagram (Figure 3). Pc < 0.5 predictsthe self-assembling into micelles, 0.5 < Pc < 1 predicts theself-association into bilayer phases which would lead to theformation of vesicles, and Pc > 1 would correspond to phaseswith negative curvature. Although the latter are not predictedin Israelachvili’s theory, those phases are compatible withinverted vesicles, as well as with hexagonal II and cubicphases (23, 31). According to this interpretation in Figure 3,within the region located at the bottom-left corner of the π-xCAd

phase space (at low xCAd and π), the resulting Pc values werecompatible with the formation of bilayers. At the bottom-rightcorner (high xCAd and low π), Pc values predicted the formationof micelles. At the top of the π-xCAd phase space, in the regionlocated above a line crossing the plane approximately alongthe diagonal from 45 mN/m at xCAd ) 0 through 28 mN/m atxCAd ) 0.4 to 18.8 at xCAd ) 1, Pc values were all above 1. Inconditions of total miscibility, this may be interpreted as anindication of a tendency to form negative curvature phases whenthe mixture is dispersed in water. However, within regions ofthe phase diagram where components are partially miscible withone another, these calculated Pc values become meaningless.Hence, to obtain stable planar membranes at a π compatiblewith the equilibrium surface pressure of biomembranes (near30-35 mN/m) the molar faction of CAd should not be higherthan 0.2.

The analysis of the variation of the Mma (mean moleculararea) or the ∆V.A (surface potential per unit of molecular surfacedensity) with the mole fraction (xCAd) at constant packingconditions (π ) 6, 18.5, or 35 mN/m) is shown in Figure 4.Straight dotted lines joined the values corresponding to themolecular parameters of pure dpPC and pure CAd. Experimentalpoints lying on this line would represent either ideal mixing orimmiscibility behavior of components in the monolayer. Thecompositional dependence or independence, respectively, of thecollapse pressure might help to discriminate between bothbehaviors. At 6 mN/m, Mma (Figure 4a) as well as ∆V.A (Figure4b) decreased as a function of xCAd. At this lateral pressure,these parameters showed positive deviations from ideality,suggesting repulsive interactions or other steric restrictions to

Figure 3. Surface pressure-composition phase diagram overlapped withcritical packing parameter (Pc) for dpPC-CAd mixtures. Numbers onthe graph refer to Pc values calculated for each mixture at the specifiedsurface pressure. NT: no structure predicted in the theory (Pc > 1).The phase diagram (π-xCAd) was constructed using πc values (9),typical pure dpPC bidimensional phase transition pressure (b), andphase transitions pressures in the mixtures (1 and O), taken from Figure2. The inset shows the Mma corresponding to those surface pressuresat which bidimensional phase transitions and collapses occur, at eachof the CAd molar fractions studied. (b) bidimensional phase transitioncharacteristic of the pure dpPC, (O and 1) other bidimensional phasetransitions observed in mixtures, and (9) Mma at the monolayer collapsepoint. Note that Pc values above the line joining the phase transitionpoints, particularly in the range 0.2 < xCAd < 0.4, might be meaninglessdue to possible immiscibility.


the ideal molecular packing between the monolayer components.While the expansion observed in Mma was very important, inthe case of ∆V.A the expansion was less significant. Theseresults indicate that the permanence of CAd at high proportionsin the monolayer at low π disrupts the organization of thephospholipids (Mma is highly expanded) without acquiring acoherent organization (changes in ∆V.A were small). A similarbehavior was in the Mma-xCAd plot at 18.5 mN/m (surfacepressure higher but still within the miscibility region accordingto the phase diagram); however, no deviation from ideality wasobserved in ∆V.A (Figure 4c,d). At 35 mN/m, the monolayerphase exists up to xCAd ) 0.6 and at higher proportions of CAda discontinuity was shown in Mma (there are no data at theseπ and xCAd, as shown in Figure 2b). Hence, at 35 mN/m, Mmaand ∆V.A vs xCAd plots decreased up to the ordinate axis andreach zero. Negligible xCAd-dependent expansions were observedwith respect to the ideality line not only in ∆V.A but also inMma (Figure 4e,f). Either ideal mixing or total immiscibilitymay have exhibited this behavior. The inspection of the phasediagram suggested immiscibility within the range 0.2 < xCAd

< 0.4 and partial miscibility within 0.4 < xCAd < 0.6 due tothe compositional constancy and dependency of πc, respectively.Contrary to what happened within 0 < xCAd < 0.1, where wefound CAd solubility in dpPC, the latter should be ascribed todpPC mixed within a continuous CAd phase. Beyond xCAd )0.6, this miscibility continued with πc decreasing up to 18.8mN/m at xCAd ) 1.

3.3.4. CAd Penetration in Phospholipid Monolayers. Theability of CAd to penetrate in the monomolecular layers of dpPCfrom the aqueous subphase was evidenced by the π increase atconstant area at different initial surface pressures up to a πcutoff

) 13.08 ( 4.6 mN/m. This value was determined by extrapolat-ing the plot of ∆π versus πi to ∆π ) 0 (Figure 5). On the otherhand, the smooth variation of the monolayer compressibility(K) with the xCAd also showed that CAd was stable in the filmat low molecular packings (low π). Above 13 mN/m, not onlydid CAd not penetrate in the film from the subphase (∆π ) 0),but also K at high π (Figure 2c) suffered a discontinuousvariation with xCAd at xCAd > 0.2. Taken together, these resultssuggest that it was not possible to stabilize high amounts ofCAd in highly packed monolayers independently of the directionfrom which the drug got access to the model membrane.However, small amounts of CAd can remain in the monolayer

at lateral surface pressures compatible with the equilibriumpressure of bilayerssca. 30-35 mN/m (32)sas shown in thephase diagram (Figure 3) as well as by the resultant dipolarcontributions at xCAd < 0.2 (Figure 2f).

CAd was unstable at intermediate xCAd (0.2-0.4) leading toimmiscibility behavior with dpPC, which at high xCAd (>0.4)became a phase inversion with dpPC partially miscible in acontinuous CAd phase. This interpretation was strongly sug-gested by the linear A-xCAd and ∆V.A-xCAd plots (Figure 4e,f)in conjunction with πc independence (within 0.2 < xCAd < 0.4)or dependence (within 0.4 < xCAd e 1) on xCAd, respectively(Figure 3).

4. CONCLUSIONS

The CAd synthesized in the present work was an amphipathicCNZ derivative capable of binding at the BZD site of theGABAA-R by means of its hydrophilic end and anchoring in a

Figure 4. Mean molecular area (a, c, and e) and surface potential per unit of molecular surface density (b, d, and f) vs composition plots at constantsurface pressures. π ) 6 (a,b), 18.5 (c,d), or 35 mN/m (e,f). Straight lines represent the behavior expected for ideal mixing or complete segregationof the components. Deviations from this behavior, particularly at 6 mN/m, are more clearly reflected in Mma-x plots.

Figure 5. Penetration of CAd in monomolecular layers of dpPC atdifferent initial molecular packings. The line represents the fitness ofa straight line to the experimental points by regression analysis by theleast-squares method. The πcutoff value, indicated by the arrow, representsthe maximum π allowing drug penetration and monolayer deformation.The regression line is defined by the equation ∆π ) a + b × πi, wherea is the ordinate (17 ( 4 mN/m) and b the slope (-1.3 ( 0.6 mN/m).At ∆π ) 0, πi equals the πcutoff ) 13.08 ( 4.6 mN/m.


biomembrane through the hydrophobic end represented by ahydrocarbon chain attached at the N1 position. The design ofthe BZD derivate was supported by theoretical studies aboutthe BZD structure-activity relationship where high-affinityanalogues interacted with three GABAA-R electrophilic groupslocated at C7, C2, and the iminic nitrogen N4. It is known thatthe N4 interaction is facilitated by the presence of halogens inC2 and when the phenyl ring is rotated in the greater coplanaritydirection between the phenyl and the plane C′1 - C5 ) N4(33). Consequently, some chemical alteration in the N1 positionwas not expected to affect (at least chemically) the BZD capacityto interact with the receptor binding site, and our resultsconfirmed this hypothesis.

Molecular parameters as well as conditions for self-assemblyin water determined for CAd were summarized in Table 1together with those of dpPC, which was taken as a reference.

CAd as a Precursor to Obtain a Probe to Evaluate theMolecular Organization of Synaptosomal Membranes, Inthe Vicinity of GABAA-R. The present findings suggested thatCAd could be stabilized at the membrane-water interface withits CNZ moiety stacked at the GABAA-R, while its methyleneend inserted within the membrane depth may be useful forsensing the molecular order in the receptor surroundingsprovided a hydrophobic label is attached to it. Hence, such achemical modification would make CAd a suitable molecularprobe. The longer the hydrocarbon chain attached at N1 positionof CNZ, the deeper the membrane regions that could be sensedwould be. It is important to recall that a probe should provideinformation about the system properties without affecting them.That is why they are used at proportions sufficiently low (e.g.,0.5 mol % representing a xprobe ) 0.005). Our results indicatethat this condition would be satisfied with CAd in bilayers whichhave an equilibrium surface pressure centered at 35 mN/m (32).

CAd as a Precursor of an Affinity-Based PurificationSystem for GABAA-R. Both the surface activity of CAd andits ability to interact with the BZD binding site at GABAA-Rare equally important characteristics that would make CAd anappropriate molecule to develop an affinity-based precipitationmethod to purify GABAA-R based on CAd coated micropar-ticles. The latter may be proposed as a useful substitute oftraditional inmunoprecipitation procedures to avoid the require-ment of monoclonal antibodies against specific epitopes ofGABAA-R subunits (34).

ACKNOWLEDGMENT

The present work was partially financed by grants fromCONICET, SECyT-Universidad Nacional de Cordoba andANPCyT from Argentina. B.C. is a Ph.D. student of theDoctorado en Ciencias Biologicas of the Universidad Nacionalde Cordoba. A.V.T. and B.C. are fellowship holders and E.L.M,G.I.Y., and M.A.P are Career Investigators from CONICET.

Supporting Information Available: All figures as well as1H NMR and 13C NMR spectra. This material is available freeof charge via the Internet at http://pubs.acs.org.

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Table 1. Physicochemical Characteristics of CAd Compared withdpPC, Both at Their Respective Collapse Points

property (units) dpPC CAd

πc (mN/m) 55.5 18.8Mma (Å2/molec) 40 49∆Vπc (V) 0.530 0.296µ⊥ (mD) a 562 385Kc (mN/m) 198 55Pc

c 0.86 0.41predicted self-organization bilayer micellexmax for 0.5 < Pc < 1 at π ) 30 mN/mb - 0.3πcutoff (mN/m) - 13

a Calculated as indicated in section 3.3.2. b Molar fraction to allowself-assembly into bilayers at a surface pressure of 30 mN/m.c Calculated at the πc of CAd.


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